IIIT Hyderabad Publications |
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A Computational Study of Protein-DNA Binding during DNA Damage Repair and Gene ActivationAuthor: Abhinandan Panigrahi Date: 2020-01-31 Report no: IIIT/TH/2020/9 Advisor:Marimuthu Krishnan AbstractUnderstanding the myriad interactions between protein and DNA that are present at the heart of most important biological phenomena is of critical importance to understanding life itself. Molecular Dynamics (MD) simulations provide a detailed view of the conformational changes that drive these intricate, multi-stage, highly dynamical protein-DNA binding processes. Aided by MD, the present work aims to investigate two processes that DNA-binding proteins take part in: (a) recognizing DNA mismatch lesions to commence the repair mechanism, and (b) regulating and affecting the process of transcription. The ultraviolet (UV) radiation-induced DNA lesions play a causal role in many prevalent genetic skin-related diseases and cancers. The damage sensing protein Rad4/XPC specifically recognizes and repairs these lesions with high fidelity and safeguards genome integrity. Despite considerable progress, the mechanistic details of the mode of action of Rad4/XPC in damage recognition remain obscure. The present thesis investigates the mechanism, energetics, dynamics, and the molecular basis for the sequence specificity of mismatch recognition by Rad4/XPC. We dissect one of the key molecular events that occur as Rad4/XPC tries to recognize and bind to DNA lesions/mismatches: the flipping of a pair of nucleotide bases at the damage/mismatch site. Using suitable reaction coordinates, the free energy surfaces for the base flipping event were determined using molecular dynamics (MD) and umbrella sampling simulations on three mismatched (CCC/CCC, TTT/TTT and TAT/TAT mismatches) Rad4-DNA complexes. We identify the key determinants of the sequence-dependent specificity of Rad4 for the mismatches and explores the ramifications of specificity in the process of base flipping. The results unravel the molecular basis for the high specificity of Rad4 towards CCC/CCC mismatch and lower specificity for the TAT/TAT mismatch. The interplay of the conformational flexibility of mismatched bases and the Rad4 specificity explored here complement recent experimental FRET studies on Rad4-DNA complexes. The cAMP-mediated allosteric transition in the catabolite activator protein (CAP) allows it to position its recognition F-helices to bind to the promoter region on DNA, resulting in gene activation. The present study aims to provide a molecular basis for the conformational changes observed during the allosteric regulation of CAP by cAMP and the subsequent binding to the DNA promoter region using MD simulation and umbrella sampling techniques. In particular, we investigate DNA binding-induced changes in the orientations and dynamics of functionally-relevant F-helices in the DNA binding domain. The two-dimensional free energy profiles of the cAMP-liganded CAP and the cAMP-CAP-DNA complex provide a clear, simple picture of the flexibility constraints imposed on the DNA binding region of CAP upon DNA-binding. We further relate these differences in the free energy surface of CAP to the dynamical modes of the recognition F-helices. The observed dynamical changes are then explained by dissecting the key interactions between CAP, DNA, and the cAMP molecules. Full thesis: pdf Centre for Computational Natural Sciences and Bioinformatics |
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